Nucleic acid analysis has played an important role for the detection of pathogens and genetic diseases. In recent years, its usefulness has been seen in many decentralized applications such as point-of-care diagnostics, environmental and food monitoring, and the detection of biological warfare agents.
In a typical electrochemical DNA detection scheme, one of the essential steps is to immobilize a DNA or PNA (peptide nucleic acid) probe on the surface of a substrate (usually the detecting electrode). In cooperation with some washing steps, the immobilization of the DNA or PNA probe enables separation and differentiation of the target DNA which hybridizes with the immobilized probe from the non-target DNAs which do not hybridize with the probe. However, problems with these “immobilization-based” electrochemical DNA detection methods arise including that the bonding strength of the surface-linked DNA might not survive the high temperature cycling during polymerase chain reaction (PCR). Furthermore, for immobilization-based electrochemical DNA detection schemes, multiplexing would be a huge challenge, as it would add a lot to complexity of the assay and the accuracy of detection may be affected by interference between the detection of different targets.
The inventors have previously developed an “immobilization-free” electrochemical DNA detection approach and disclosed in Luo, X., et al. (Anal. Chem. 2008). This approach uses ferrocene-labeled PNA based on the neutral PNA backbone and the electrostatic interaction between the negative DNA backbone and the negative electrode surface. The inventors have also showed the success in using this approach for multiplexed DNA detection and disclosed in Luo X., et al. (Biosens. and Bioelectron. 2009). Apart from the detection strategy discussed above, other electrochemical DNA detection schemes that require no probe immobilization with “signal-off” operation have also been studied and reported by other groups. For instance, Tamiya et al. reported in their papers (Ahmed M. U., et al., Analyst 2007; Kobayashi M., et al., Electrochem. Commun. 2004) such an immobilization-free DNA detection method based on the reduced diffusion coefficient of intercalated Hoechst 33258 leading to a reduced electrochemical signal in the presence of double stranded DNA. A similar strategy has also been developed by Gong's group, using methylene blue for the real-time electrochemical monitoring of PCR amplicons as disclosed in Fang T. H., et al. (Biosens. and Bioelectron. 2009).
In addition, the principle of “competitive hybridization” has been utilized for electrochemical DNA detection in a number of reports, such as Duwensee H., et al. (Analyst 2009), Kim K., et al. (Chem. Commun. 2004), Liepold P., et al. (Anal. Bioanal. Chem. 2008), and Mir M., et al. (Anal. Bioanal. Chem. 2008). All these detection schemes have one characteristic in common, which is that the competitive hybridization occurs at the interface between a solution and an electrode surface. As the DNA probe is immobilized on the electrode, the target DNA and the competitor DNA has to diffuse to the electrode surface in order to hybridize with the probe, which results in low hybridization efficiency and therefore, long assay time.
Among the available analytical techniques for DNA analyses, real-time polymerase chain reaction has been a key technology for high-speed testing and accurate quantification.
Various assays based on real-time PCR have been developed utilizing fluorescence-linked reporters such as SYBR Green 1, hydrolysis probe, and hybridization probes for simultaneous deoxyribonucleic acid (DNA) amplification and PCR amplicon detection. Despite wide acceptance, their use is largely limited in clinical and research laboratory settings. The difficulty in advancing this technology for point-of-care testing (POCT) applications lies in the requirement of bulky and complex optical systems for the DNA amplicon detection. The goal of performing complete DNA analyses with a hand-held instrument is not attainable based on optical detection systems that are bulky and cumbersome. A far more suitable alternative for this type of use and one that is extremely suited for POCT, is a system based on the detection of electrochemical signals.
Over the past years, numerous studies have been carried out on electrochemical DNA sensors, some of which focused on PCR amplicon detection. Efforts have also been made in developing DNA microchips having an attached electrochemical signaling label employed in conjunction with an electrochemical detection system for post-PCR product identification. The latter prior art post-PCR hybridization-based platform suffers from a long assay time and has a narrow dynamic range when compared to fluorescence-based real-time PCR methods.
In view thereof, there is a need for developing a method for detecting and quantifying nucleic acid(s) in a sample that is accurate, reproducible, and safe and, at the same time, may be performed in small scale devices.